U.S. patent application number 16/121606 was filed with the patent office on 2018-12-20 for ovarian cancer specifically targeted biodegradable amphiphilic polymer, polymer vesicle prepared thereby and use thereof.
This patent application is currently assigned to BRIGHTGENE BIO-MEDICAL TECHNOLOGY CO., LTD.. The applicant listed for this patent is BRIGHTGENE BIO-MEDICAL TECHNOLOGY CO., LTD.. Invention is credited to FENGHUA MENG, JIANDONG YUAN, ZHIYUAN ZHONG, YAN ZOU.
Application Number | 20180360766 16/121606 |
Document ID | / |
Family ID | 56307950 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180360766 |
Kind Code |
A1 |
YUAN; JIANDONG ; et
al. |
December 20, 2018 |
OVARIAN CANCER SPECIFICALLY TARGETED BIODEGRADABLE AMPHIPHILIC
POLYMER, POLYMER VESICLE PREPARED THEREBY AND USE THEREOF
Abstract
Provided are an ovarian cancer specifically targeted
biodegradable amphiphilic polymer, a polymer vesicle prepared
thereby and use thereof. The biodegradable amphiphilic polymer is
prepared by a polymer containing dithiocarbonate monomer bonded
with targeting molecules. The polymer vesicle prepared with the
biodegradable amphiphilic polymer can crosslink spontaneously
without adding an extra crosslinker, and the crosslink has a
reduction activity and hence can be used in drug controlled-release
systems, and contributes to the clinical use and production of
nano-drugs.
Inventors: |
YUAN; JIANDONG; (SUZHOU,
CN) ; MENG; FENGHUA; (SUZHOU, CN) ; ZOU;
YAN; (SUZHOU, CN) ; ZHONG; ZHIYUAN; (SUZHOU,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRIGHTGENE BIO-MEDICAL TECHNOLOGY CO., LTD. |
Suzhou |
|
CN |
|
|
Assignee: |
BRIGHTGENE BIO-MEDICAL TECHNOLOGY
CO., LTD.
|
Family ID: |
56307950 |
Appl. No.: |
16/121606 |
Filed: |
September 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/CN2017/075529 |
Mar 3, 2017 |
|
|
|
16121606 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 5/00 20130101; C08G
64/18 20130101; C08G 64/30 20130101; A61K 47/551 20170801; A61K
9/5146 20130101; A61K 47/6855 20170801; A61K 47/6935 20170801; A61K
47/62 20170801; A61P 35/00 20180101; C08G 63/688 20130101; C08G
63/64 20130101; A61K 9/5169 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61P 35/00 20060101 A61P035/00; C08G 64/18 20060101
C08G064/18; C08G 64/30 20060101 C08G064/30 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2016 |
CN |
201610123977.1 |
Claims
1. An ovarian cancer specifically targeted biodegradable
amphiphilic polymer, wherein the ovarian cancer specifically
targeted biodegradable amphiphilic polymer is prepared by bonding a
polymer containing dithiocarbonate monomer with a targeting
molecule; wherein the targeting molecule is GE11 polypeptide, folic
acid FA, transferrin or Herceptin protein; and wherein the polymer
containing dithiocarbonate monomer has a chemical structure of
Formula I or Formula II:: ##STR00015## wherein R1 is selected from
one of the following groups: ##STR00016## R2 is selected from one
of the following groups: ##STR00017## and wherein k is from 113 to
170, x is from 15 to 45, y is from 80 to 300, and m is from 220 to
280, and wherein the polymer containing dithiocarbonate monomer has
a molecular weight of 30 kDa to 55 kDa when it has a chemical
structure of the Formula I, and has a molecular weight of 60 kDa to
95 kDa when it has a chemical structure of the Formula II.
2. A polymeric vesicle, wherein the polymeric vesicle is prepared
by one of the following preparation methods: (1) prepared by the
ovarian cancer specifically targeted biodegradable amphiphilic
polymer according to claim 1; (2) prepared by the polymer
containing dithiocarbonate monomer as mentioned in claim 1; (3)
prepared by the ovarian cancer specifically targeted biodegradable
amphiphilic polymer and polymer containing dithiocarbonate monomer
according to claim 1; (4) prepared by coupling a targeting molecule
to the surface of the polymeric vesicles prepared by the polymer
containing dithiocarbonate monomer as mentioned in claim 1, wherein
the targeting molecule is GE11 polypeptide, folic acid, transferrin
or Herceptin protein.
3. The polymeric vesicle according to claim 2, wherein the
polymeric vesicle is a self-crosslinked polymeric vesicle, and the
self-crosslinked polymeric vesicle has a particle size of 50 nm to
160 nm.
4. The polymeric vesicle according to claim 2, wherein the
polymeric vesicle is prepared by the ovarian cancer specifically
targeted biodegradable amphiphilic polymer and the polymer
containing dithiocarbonate monomer according to claim 1; and
calculated according to percentage of mass, the ovarian cancer
specifically targeted biodegradable amphiphilic polymer is in an
amount of 1 wt. % to 40 wt. % by mass.
5. A method for preparing a drug for treating ovarian cancer,
wherein the polymeric vesicle according to claim 2 is used as a
carrier of the drug.
6. The method according to claim 5, wherein the drug for treating
ovarian cancer is a small molecular anticancer drug.
7. The method according to claim 6, wherein the small molecular
anticancer drug is paclitaxel, docetaxel, adriamycin, olaparib,
gefitinib, doxorubicin hydrochloride, epirubicin hydrochloride, or
irinotecan hydrochloride.
8. A method for preparing a nanomedicine for treating ovarian
cancer, wherein the nanomedicine is prepared with the ovarian
cancer specifically targeted biodegradable amphiphilic polymer
according to claim 1.
9. A method for preparing a nanomedicine for treating ovarian
cancer, wherein the nanomedicine is prepared with the polymeric
vesicle according to claim 2.
10. A method for preparing a drug for treating ovarian cancer,
wherein the polymeric vesicle according to claim 3 is used as a
carrier of the drug.
11. A method for preparing a drug for treating ovarian cancer,
wherein the polymeric vesicle according to claim 4 is used as a
carrier of the drug.
12. The method according to claim 10, wherein the drug for treating
ovarian cancer is a small molecular anticancer drug.
13. The method according to claim 11, wherein the drug for treating
ovarian cancer is a small molecular anticancer drug.
14. The method according to claim 12, wherein the small molecular
anticancer drug is paclitaxel, docetaxel, adriamycin, olaparib,
gefitinib, doxorubicin hydrochloride, epirubicin hydrochloride, or
irinotecan hydrochloride.
15. The method according to claim 13, wherein the small molecular
anticancer drug is paclitaxel, docetaxel, adriamycin, olaparib,
gefitinib, doxorubicin hydrochloride, epirubicin hydrochloride, or
irinotecan hydrochloride.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/CN2017/075529, filed on Mar. 3, 2017, which is
based upon and claims priority to Chinese Patent Application No.
201610123977.1, filed on Mar. 4, 2016, and the entire contents of
which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a biodegradable polymer
material and application thereof, in particular, to an ovarian
cancer specifically targeted biodegradable amphiphilic polymer, a
polymeric vesicle prepared thereby and application thereof in the
targeted therapy of ovarian cancer. The present disclosure belongs
to the field of medical materials.
BACKGROUND
[0003] Biodegradable polymers have very unique properties and are
widely applied in various fields of biomedicine, such as surgical
sutures, bone fixation devices, scaffold materials for biological
tissue engineering, and carriers for controlled drug release.
Synthetic biodegradable polymers mainly include aliphatic
polyesters (polyglycolide PGA, polylactide PLA, lactide-glycolide
copolymer PLGA, polycaprolactone PCL), polycarbonate
(Polytrimethylene carbonate, PTMC), which are the most commonly
used biodegradable polymer and have been approved by the US Food
and Drug Administration (FDA).
[0004] However, the existing biodegradable polymers such as PTMC,
PCL, PLA, and PLGA have relatively simple structures and lack
modifiable functional groups, as a result it is always difficult to
provide a drug carrier for stable circulation. The degradation
products of polycarbonate are mainly carbon dioxide and neutral
diols, and do not produce acidic degradation products. The
functional cyclic carbonate monomer can be copolymerized with
cyclic ester monomers such as GA, LA and .epsilon.-CL etc., as well
as other cyclic carbonate monomers to obtain biodegradable polymers
with different properties.
[0005] In addition, the biodegradable nano-carrier prepared by the
biodegradable polymer of the prior art has the problems of unstable
circulation in the body, low uptake for tumor cells, and low drug
concentration within cells, which lead to low efficacy of the
nanomedicine and toxic side effects. The micelle nanomedicine
prepared by functional biodegradable polymers maintains circulation
stability in the body, but can only be loaded with hydrophobic
small molecular anticancer drugs other than the hydrophilic small
molecular anticancer drugs with stronger penetrability, which
limits it use as a drug carrier.
[0006] Cancer is a main threaten to human health, and its morbidity
and mortality are increasing year by year. Ovarian cancer is a
malignant tumor of ovarian, which refers to a malignant tumor that
grows on the ovarian. 90% to 95% of the ovarian cancer is primary
ovarian cancer, and the remaining 5% to 10% of the ovarian cancer
is due to the transfer of primary cancer from other parts. Due to
the complexity of embryonic development, tissue anatomy and
endocrine function for the ovarian, the ovarian tumor can be benign
or malignant. Due to the lack of symptoms of specificity in early
ovarian cancer and the limited effects of screening, it is very
difficult to identify the tissue type of the ovarian tumor and
whether it is benign and malignant. Therefore, it is difficult to
perform early diagnosis. The tumor confined to the ovarian is found
to only account for 30% during laparotomy of ovarian cancer. Most
of cases show that the tumor has spread to the bilateral
attachments of the uterus, the omentum majus and organs in pelvic
cavity. 60% to 70% of the patients are diagnosed as advanced
cancers that have poor therapeutic effects. Until now, it is
difficult to diagnose and treat ovarian cancer. Therefore, although
the incidence of ovarian cancer is lower than that of cervical
cancer and endometrial cancer, and the incidence is in the third
position of the gynecological malignancies, the mortality rate
exceeds the sum of cervical cancer and endometrial cancer, which is
the highest in gynecological cancer, thereby the ovarian cancer is
a serious threat to the health of women.
[0007] In short, ovarian cancer is a female malignant tumor with
high incidence. Although the absolute number of cases is not that
many, the mortality rate is very high mainly because it is
difficult to detect and diagnose at early stage. Most of patients
are diagnosed as advanced and thus miss the best time for resection
surgery. Further there are many other characteristics, such as low
cure rate, easy to transfer and easy to have drug resistance during
the therapy of the ovarian cancer. Compared with traditional
chemotherapeutic drugs, the distribution in vivo of nanomedicines
can be changed, thereby increasing the concentration of drugs in
tumors and improving the therapeutic effects. Nanomedicine is a key
point and is the hope for the treatment of ovarian cancer. DOXIL
(PEGylated liposomal adriamycin) is a liposome vesicle nano drug
DOXIL which is approved firstly by FDA and is clinically effective
for treating ovarian cancer. However, there are some problems with
DOXIL. Firstly the maximum tolerated dose (MTD) is small, so the
treatment window is relatively narrow, and it is easy to have toxic
side effects. Secondly, DOXIL is based on the passive targeting
function of EPR effect, thus itis difficult to transport
nanomedicine to all tumor tissues and cells using a common unified
mechanism due to the huge individual differences among different
tumors. (See: S. Eetezadi, S N. Ekdawi, C. Allen, Adv. Drug Deliv
Rev, 2015, 91, 7-22). For different tumors, personalized treatment
is particularly important because the surface properties of
different tumors are very different, and the same tumors are also
very different among different patients; even the tumor cells in
the same tumor are different. Therefore, it is necessary to
personally design a targeting system suitable for the target tumor,
and the drug suitable for one indication cannot be used in other
diseases, thus the personalized treatment is particularly
important. Therefore, it is necessary to develop an active
targeting nano drug against a specific tumor, which has tumor
specificity to realize other advantages of the nano drug, for
example increasing the effective drug concentration for the tumor
cell and improving the therapeutic effect in vitro and in vivo.
SUMMARY
[0008] The object of the present invention is to provide an ovarian
cancer specifically targeted biodegradable amphiphilic polymer, a
polymeric vesicle prepared therefrom, and application thereof as a
carrier for an anti-ovarian cancer drug in preparation of
ovarian-targeting therapeutic drugs.
[0009] The above object is achieved through the following technical
solutions:
[0010] An ovarian cancer specifically targeted biodegradable
amphiphilic polymer, which is prepared by bonding a polymer
containing dithiocarbonate monomer with a targeting molecule;
wherein the targeting molecule is GE11 polypeptide, folic acid FA,
transferrin or Herceptin protein; and wherein the polymer
containing dithiocarbonate monomer has a chemical structure of one
of the following formulas:
##STR00001##
wherein R1 is selected from one of the following groups:
##STR00002##
[0011] R2 is selected from one of the following groups:
##STR00003##
wherein k is from 113 to 170, x is from 15 to 45, y is from 80 to
300, and m is from 220 to 280.
[0012] The polymer containing dithiocarbonate monomer disclosed in
the present invention has a hydrophobic block that contains a
cyclic carbonate unit containing a double-sulfur five-membered ring
functional group in the side chain. the polymer containing
dithiocarbonate monomer can be a diblock polymer as below:
##STR00004##
or a triblock polymer as below:
##STR00005##
[0013] In a preferred embodiment, R1 is selected from one of the
following groups:
##STR00006##
[0014] R2 is selected from one of the following groups:
##STR00007##
[0015] K is 113 to 170, x is 20 to 40, and y is 125 to 250.
[0016] Preferably, the polymer containing dithiocarbonate monomer
has a molecular weight of 30 kDa to 55 kDa when it has a chemical
structure of the Formula I, and has a molecular weight of 60 kDa to
95 kDa when it has a chemical structure of the Formula II. The
molecular weight of the polymer disclosed in the present invention
is controllable, and the constitution and ratio of each structural
unit are suitable for self-crosslinked to form a stable polymeric
vesicle structure.
[0017] The ovarian cancer specifically targeted biodegradable
amphiphilic polymer disclosed in the invention has
biodegradability, and its hydrophobic portion has a molecular
weight which is about three times or more than three times of the
hydrophilic portion. The polymeric vesicle structure can be
prepared by a solvent displacement method, a dialysis method or a
filming-rehydration method et al. The prepared polymeric vesicle is
of nano-size and has a particle size of 50-160 nm, and can be used
as a carrier for drugs for treating ovarian cancer. A hydrophobic
small molecule anti-ovarian cancer drug such as paclitaxel,
docetaxel, adriamycin, olaparib, and gefitinib, etc. can be loaded
in a hydrophobic membrane of the vesicle, and a hydrophilic
anti-ovarian cancer drug, especially hydrophilic small molecule
anticancer drugs such as doxorubicin hydrochloride, epirubicin
hydrochloride, or irinotecan hydrochloride or mitoxantrone
hydrochloride, can be loaded in a large hydrophilic inner cavity of
the vesicle, thereby overcoming the defect that the existing
micellar carrier formed from the existing amphiphilic polymer are
only applicable for loading hydrophobic drug and the defect that in
the prior art there is no carrier which can be efficiently loaded
with the hydrophilic small molecular anticancer drugs and be stable
in vivo circulation.
[0018] The present invention also discloses a polymeric vesicle
which is prepared by the above-mentioned polymer containing
dithiocarbonate monomer, or prepared by the above-mentioned ovarian
cancer specifically targeted biodegradable amphiphilic polymer, or
prepared by the above mentioned polymer containing dithiocarbonate
monomer and the ovarian cancer specifically targeted biodegradable
amphiphilic polymer. For example, by mixing the above-mentioned
polymer containing dithiocarbonate monomer with the ovarian cancer
specifically targeted biodegradable amphiphilic polymer at
different ratios, polymeric vesicles with different targeting
densities can be prepared, i.e. obtaining ovarian cancer targeted
self-crosslinked polymeric vesicles, thereby increasing the uptake
of the polymeric vesicle nanomedicine in ovarian cancer cells. For
another example, ovarian cancer targeted polymeric vesicles can
also be prepared by coupling a tumor cell specifically targeted
molecule to an outer surface of the polymeric vesicle prepared from
the polymer containing dithiocarbonate monomer, to increase the
uptake in ovarian cancer cells. For example, PEG end of the
polymeric vesicle can be bonded to GE11, FA, transferrin or
Herceptin, et al. by Michael addition or amidation reaction.
Preferably, the polymeric vesicle of the present invention is
prepared by the ovarian cancer specifically targeted biodegradable
amphiphilic polymer in an amount of 1 to 40 wt. % by mass and the
polymer containing dithiocarbonate monomer.
[0019] The polymer containing dithiocarbonate monomer and the
ovarian cancer specifically targeted biodegradable amphiphilic
polymer of the present invention can be crosslinked with each other
without adding any substance, to obtain a self-crosslinked
polymeric vesicle. When the polymer is applied as a drug carrier,
the most basic and critical property for achieving optimal
targeting and therapeutic effects is the long time circulation in
vivo. The formation of the cross-linked structure is a necessary
process for the polymer carrier to circulate in vivo for long time.
In the prior art, a stable cross-linked structure of the polymer
nano-carrier is formed by adding a crosslinking agent, but the
addition of the crosslinking agent can not only increase the
complexity for preparing nanomedicines, increase the production
cost of nanomedicine, and reduce the final purity of the drug,
which is not conducive to the amplification production for clinical
application of nanomedicine, but also affect loading efficiency of
the drug and drug release level, increase toxic side effects and
reduce the biocompatibility of the nano drug loaded in the polymer
carrier. The polymer structure firstly disclosed in the present
invention can be self-crosslinked without the addition of a
crosslinking agent, thereby forming a stable chemical cross-linked
structure inside a hydrophobic film in the vesicle. As a result,
the polymer can circulate stably in vivo for long time. Since no
crosslinking agent is added, side effects due to the crosslinking
agent are avoided. Furthermore, the drug loaded polymeric vesicles
can be decrosslinked quickly in the presence of a large amount of
reducing substances within the cell, thereby releasing the maximum
amount of drug to kill the ovarian cancer cells after the drug
loaded polymeric vesicles reach the tumor and are endocytosed into
the cancer cells. Meanwhile, the stability of self-crosslinked
polymers equals to or even better than that of the cross-linked
polymeric vesicles prepared by crosslinking agent. More
importantly, in the present invention, interference of the
crosslinking agent to some drugs is avoided. By using the
self-crosslinked polymeric vesicles to load drugs, side effects of
existing small molecule drugs are avoided, and the application
range of the anticancer drug is broadened, and moreover, it can be
applied to different individuals with big physiological
differences.
[0020] The present invention also discloses the use of the ovarian
cancer specifically targeted biodegradable amphiphilic polymer in
preparation of a nanomedicine for treating ovarian cancer. Further,
the present invention also discloses the use of the above polymeric
vesicle in preparation of a nanomedicine for treating ovarian
cancer; in particular the use of the self-crosslinked polymer
vesicle as a carrier in the preparation of a drug for treating
ovarian cancer. Due to the use of self-crosslinked polymeric
vesicles, it is avoided to use crosslinking agents, which further
enhances drug safety and reduces the drug assembly steps. The
anti-ovarian cancer nanomedicine prepared based on the polymer of
the present invention is a vesicle anti-ovarian cancer
nanomedicine.
[0021] Based on the implementation of the above technical solution,
the present invention has the following advantages compared with
the prior art:
[0022] 1. The biodegradable amphiphilic polymer with a side chain
containing double-sulfur disclosed in the invention has
biodegradability and excellent targeting property for ovarian
cancer, which can be used to prepare polymeric vesicles for loading
drugs of different properties, and can be self-crosslinked to form
a stable self-crosslinked polymeric vesicle for the nanomedicine
without adding any crosslinking agent, thereby overcoming the
defects that nanomedicines in prior art are instable during
circulation in vivo and easy to release drugs too early to result
in toxic side effects.
[0023] 2. The crosslinking of the self-crosslinked polymeric
vesicle for the nanomedicine disclosed by the invention is
reversible, i.e. it supports long circulation in vivo and can be
highly enriched in ovarian cancer cells. However, it can be quickly
decrosslinked after entering the ovarian cancer cells and release
the drug to kill ovarian cancer cells with high-efficiency and
specificity without toxic and side effects, and this overcomes the
defects that the crosslinked nanomedicine in the prior art is too
stable, and the drug release in the cell is slow, which results in
drug resistance.
[0024] 3. The ovarian cancer specifically targeted biodegradable
amphiphilic polymer disclosed in the present invention can be used
to prepare self-crosslinked polymeric vesicles without adding any
crosslinking agent. The preparation method is simple, thereby
overcoming the defects that it is necessary to add substances such
as crosslinking agents in preparation of cross-linked nanomedicines
in the prior art and the defects that complicated operations and
purification processes are required. Therefore this is beneficial
to the clinical application of nanomedicines.
[0025] 4. The self-crosslinked polymeric vesicle prepared by the
self-assembly of the ovarian cancer specifically targeted
biodegradable amphiphilic polymer disclosed in the invention can be
used in the controlled release system of hydrophilic small molecule
anticancer drug, thereby overcoming defects that the existing
degradable nano-micelle carrier is only suitable for loading
hydrophobic small molecule drugs and there is no hydrophilic small
molecule anticancer drug in the prior art which can be efficiently
loaded and circulates stably. Further, self-crosslinked polymeric
vesicles targeting to the ovarian cancer can be prepared, which
have wider application in the efficient and targeted therapy of
ovarian cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a graph illustrating the distribution of the
particle size (A), the and transmission electron microscopy (B) of
the cross-linked polymeric vesicle PEG5k-P(CDC5.8k-co-TMC23k) in
the Example 15, the stability test (C) and reduction responsiveness
test (D) of the cross-linked polymeric vesicles;
[0027] FIG. 2 is a graph illustrating the in vitro release of the
DOX.HCl-loaded cross-linked polymeric vesicles
PEG5k-P(CDC5.8k-co-TMC23k) in Example 20;
[0028] FIG. 3 is a graph illustrating the in vitro release of the
DOX.HCl-loaded cross-linked polymeric vesicles GE11-CLPs in Example
20;
[0029] FIG. 4 is a graph illustrating the results of toxicity of
targeting cross-linked polymeric vesicles GE11-CLPs to SKOV3
ovarian cancer cells in Example 21;
[0030] FIG. 5 is a graph illustrating the results of toxicity of
the DOX.HCl-loaded targeted cross-linked polymeric vesicles
GE11-CLPs to SKOV3 ovarian cells in Example 22;
[0031] FIG. 6 is a graph illustrating the results of semi-lethal
toxicity of the DOX.HCl-loaded targeted cross-linked polymeric
vesicles GE11-CLPs to SKOV3 ovarian cells in Example 22;
[0032] FIG. 7 is a graph illustrating the results of endocytosis of
SKOV3 ovarian cancer cells by the DOX.HCl-loaded targeted
cross-linked polymeric vesicles GE11-CLPs in Example 24;
[0033] FIG. 8 is a graph illustrating the effects of targeting
cross-linked polymeric vesicles GE11-CLPs loaded DOX.HCl to blood
circulation in mice in Example 29;
[0034] FIG. 9 is a graph illustrating biodistribution of the
DOX-HCl-loaded targeted cross-linked vesicles GE11-CLPs in mice
carrying subcutaneous ovarian cancer in Example 30;
[0035] FIG. 10 is a graph illustrating the maximum tolerated dose
of the DOX.HCl-loaded targeted cross-linked vesicles GE11-CLPs in
mice in Example 31;
[0036] FIG. 11 is a graph illustrating a multi-dose treatment to
mice carrying subcutaneous ovarian cancer by the DOX.HCl-loaded
targeted cross-linked vesicles GE11-CLPs in Example 32, in which A
is a tumor growth curve, B is a tumor image after treatments of the
mice, C shows the change in body weight, and D is the survival
curve;
[0037] FIG. 12 is a graph illustrating a single-dose treatment to
mice carrying subcutaneous ovarian cancer by the DOX.HCl-loaded
targeted cross-linked vesicles GE11-CLPs in Example 33, in which
the A is a tumor growth curve, B is a tumor image after treatment
in mice, C is the weight change curve, and D is the survival
curve.
DETAILED DESCRIPTION
[0038] The present invention will be further described below in
combination with the examples and the accompanying drawings:
Example 1
Synthesis of a Cyclic Carbonate Monomer (CDC) which contains a
Double-Sulfur Five-Membered Ring Function Group
[0039] Sodium hydrosulfide monohydrate (28.25 g, 381.7 mmol) was
dissolved in 400 mL of N,N-dimethyl formamide (DMF) and heated to
50.degree. C. to achieve complete dissolution. Then dibromo
neopentyl glycol (20 g, 76.4 mmol) was added dropwise to carry out
reaction which lasted for 48 hours. The resultant was distilled
under a reduced pressure to remove the solvent DMF, then diluted
with 200 mL of distilled water, and extracted four times with 250
mL of ethyl acetate each time to obtain an organic phase. The
organic phase was rotary evaporated to give a yellow viscous
compound A, with a yield of 70%. The compound A was dissolved in
400 mL of tetrahydrofuran (THF) and was allowed to stand in air for
24 hours, as a result intermolecular thiol was oxidized to a
sulfur-sulfur bond to give a compound B with a yield>98%. The
compound B (11.7 g, 70.5 mmol) was dissolved in dried THF (150 mL)
and stirred until completely dissolved under nitrogen. It was then
cooled to 0.degree. C. Ethyl chloroformate (15.65 mL, 119.8 mmol)
was added and then Et.sub.3N (22.83 mL, 120.0 mmol) was added
dropwise. After the addition was completed, the system was further
reacted for 4 h under ice-water bath conditions. After the reaction
was completed, Et.sub.3N.HCl was filtered off, and the filtrate was
concentrated by rotary evaporation, then it was recrystallized for
several times by using diethyl ether to obtain a yellow crystalline
which is a cyclic carbonate monomer (CDC) that contains a
double-sulfur five-membered ring functional group, with a yield of
64%.
Example 2
Synthesis of a Diblock Polymer PEG5k-P(CDC5.8k-co-TMC23k)
[0040] Under nitrogen atmosphere, 0.1 g (0.52 mmol) of CDC monomer
and 0.4 g (4.90 mmol) of trimethylene carbonate (TMC) were
dissolved in 5 mL of dichloromethane and placed to a seal reactor,
and then 0.12 g (0.02 mmol) CH.sub.3O-PEG5000 and 0.5 mL of
catalyst solution of bi(bistrimethylsilyl)amine zinc in
dichloromethane (0.1 mol/L) were added, then the reactor was sealed
and transferred out of the glove box. After 2 days of reaction in
oil bath at 40.degree. C., the reaction was quenched with glacial
acetic acid, precipitated in iced diethyl ether, and finally
filtered and vacuum dried to give PEG5k-P(CDC 5.8k-co-TMC23k).
.sup.1H NMR (400 MHz, CDCl.sub.3): 2.08 (t,
--COCH.sub.2CH.sub.2CH.sub.2O--), 3.08 (s, --CCH.sub.2), 3.30 (m,
--OCH.sub.3), 3.65 (--OCH.sub.2CH.sub.2O--), 4.28 (t,
--COCH.sub.2CH.sub.2CH.sub.2O--), 4.31 (m, --CCH.sub.2). The
nuclear magnetic resonance was used to calculate the following:
k=114, x=30.2 and y=225.5. Molecular weight measured by GPC: 45.6
kDa, molecular weight distribution: 1.53.
##STR00008##
Example 3
Synthesis of a Diblock Polymer Mal-PEG6k-P(CDC4.8k-co-TMC19.2k)
[0041] Under nitrogen atmosphere, 0.1 g (0.52 mmol) of CDC monomer
and 0.4 g (3.85 mmol) of TMC were dissolved in 3 mL of
dichloromethane and placed to a seal reactor, and then 0.12 g (0.02
mmol) of Mal-PEG6000 and 0.1 mol/L catalyst solution of
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mol/L) were
added, then the reactor was sealed and transferred out of the glove
box. After 2 days of reaction in oil bath at 40.degree. C., the
reaction was quenched with glacial acetic acid, precipitated in
iced diethyl ether, and finally filtered and vacuum dried to give
Mal-PEG6kP(CDC4.8k-co-TMC19.2k). .sup.1H NMR (400 MHz, CDCl.sub.3):
2.08 (t, --COCH.sub.2CH.sub.2CH.sub.2O--), 3.08 (s, --CCH.sub.2),
3.30 (m, --OCH.sub.3), 3.65 (t, --OCH.sub.2CH.sub.2O--), 4.28 (t,
--COCH.sub.2CH.sub.2CH.sub.2O--), 4.31 (m, --CCH.sub.2), and 6.70
(s, Mal). The nuclear magnetic resonance was used to calculate the
following: k=136, x=25, y=188. Molecular weight measured by GPC:
38.6 kDa, molecular weight distribution: 1.42.
##STR00009##
Example 4
Synthesis of a Diblock Polymer NHS-PEG6.5k-P(CDC6k-co-TMC22.6k)
[0042] Under nitrogen atmosphere, 0.1 g (0.52 mmol) of CDC monomer
and 0.42 g (4.12 mmol) of TMC were dissolved in 5 mL of
dichloromethane and placed to a seal reactor, and then 0.11 g
(0.017 mmol) of NHS-PEG6500 and 0.5 mL of catalyst solution of
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mol/L) were
added, then the reactor was sealed and transferred out of the glove
box. After 2 days of reaction in oil bath at 40.degree. C., the
reaction was quenched with glacial acetic acid, precipitated in
iced diethyl ether, and finally filtered and vacuum dried to give
NHS-PEG 6.5k-P(CDC6k-co-TMC22.6). .sup.1H NMR (400 MHz,
CDCl.sub.3): 2.08 (t, --COCH.sub.2CH.sub.2CH.sub.2O--), 3.08 (s,
--CCH.sub.2), 3.30 (m, --OCH.sub.3), 3.65 (--OCH.sub.2CH.sub.2O--),
4.28 (t, --COCH.sub.2CH.sub.2CH.sub.2O--), 4.31 (m, --CCH.sub.2),
and 2.3 (s, NHS). The nuclear magnetic resonance was used to
calculate the following: k=148, x=31.3, and y=221.6. Molecular
weight measured by GPC: 51.3 kDa, molecular weight distribution:
1.43.
##STR00010##
Example 5
Synthesis of a Diblock Polymer PEG7.5k-P(CDC5.8k-co-TMC20.0k)
[0043] Under nitrogen atmosphere, 60 mg (0.31 mmol) of CDC monomer
and 0.2 g (1.93 mmol) of TMC were dissolved in 1 mL of
dichloromethane and placed to a seal reactor, and then 75 mg (0.01
mmol) of CH.sub.3O-PEG7500 and 0.5 mL of catalyst solution of
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mol/L) were
added, then reacted in oil bath at 40.degree. C. for 2 days.
Subsequent processing steps were the same as those in Example 2, to
obtain PEG7.5k-P(CDC5.8k-co-TMC20.0k). The reaction formula and the
characteristic peaks of .sup.1H NMR are the same as Example 2. The
nuclear magnetic resonance was used to calculate the following:
k=170, x=30, and y=196. Molecular weight measured by GPC: 54.5 kDa,
molecular weight distribution: 1.36.
Example 6
Synthesis of a Diblock Polymer PEG5k-P(CDC3.9k-co-LA18.0k)
[0044] Under nitrogen atmosphere, 0.08 g (0.42 mmol) of CDC and 0.3
g (2.1 mmol) of lactide (LA) were dissolved in 2 mL of
dichloromethane and placed to a seal reactor, and then 0.1 g (0.02
mmol) of CH.sub.3O-PEG5000 and 0.1 mol/L of catalyst
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mL) were
added, then reacted in oil bath at 40.degree. C. for 2 days, and
subsequent processing steps were the same as those in Example 2 to
obtain PEG5k-P(CDC3.9k-co-LA14.6k). .sup.1H NMR (400 MHz,
CDCl.sub.3): 1.59 (s, --COCH(CH.sub.3)O--), 3.08 (s, --CCH.sub.2),
3.30 (m, --OCH.sub.3), 3.65 (--OCH.sub.2CH.sub.2O--), 4.31 (m,
--CCH.sub.2), 5.07 (s, --COCH(CH.sub.3)O--). The nuclear magnetic
resonance was used to calculate the following: k=114, x=20, and
y=125. Molecular weight measured by GPC: 34.3 kDa, molecular weight
distribution: 1.32.
##STR00011##
Example 7
Synthesis of a Diblock Polymer PEG6.5k-P(CDC5.8k-co-LA28.3k)
[0045] Under nitrogen atmosphere, 0.1 g (0.57 mmol) of CDC and 0.5
g (3.5 mmol) of LA were dissolved in 3 mL of dichloromethane and
placed to a seal reactor, and then 0.11 g (0.015 mmol) of
CH.sub.3O-PEG6500 and 0.5 mL of catalyst solution of
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mol/L) were
added, then reacted in oil bath at 40.degree. C. for 2 days, and
subsequent processing steps were the same as those in Example 6 to
obtain PEG6.5k-P(CDC5.8k-co-LA28.3k). The nuclear magnetic
resonance was used to calculate the following: k=148, x=30, and
y=190. Molecular weight measured by GPC: 42.4 kDa, molecular weight
distribution: 1.43.
Example 8
Synthesis of a Diblock Polymer Mal-PEG6k-P(CDC3.6k-co-LA18.6k)
[0046] Under nitrogen atmosphere, 0.1 g (0.52 mmol) of CDC and 0.5
g (5.56 mmol) of LA were dissolved in 4 mL of dichloromethane, and
placed to a seal reactor, and then 0.15 g (0.025 mmol) of
Mal-PEG6000 and 0.1 mol/L of catalyst solution of
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.1 mL) were
added, then reacted in oil bath at 40.degree. C. for 2 days, and
subsequent processing steps were the same as those in Example 2 to
obtain Mal-PEG6k-P(CDC3.6k-co-LA18.6k). .sup.1H NMR (400 MHz,
CDCl.sub.3): 1.59 (s, --COCH(CH.sub.3)O--), 3.08 (s, --CCH.sub.2),
3.30 (m, --OCH.sub.3), 3.65 (t, --OCH.sub.2CH.sub.2O--), 4.31 (m,
--CCH.sub.2), 5.07 (s, --COCH(CH.sub.3)O--), and 6.70 (s, Mal). The
nuclear magnetic resonance was used to calculate the following:
k=136, x=20, and y=129. Molecular weight measured by GPC: 32.5 kDa,
molecular weight distribution: 1.44.
##STR00012##
Example 9
Synthesis of Triblock Polymer
P(CDC3.8k-co-TMC18.8k)-PEG10k-P(CDC3.8k-co-TMC18.8k)
[0047] Under nitrogen atmosphere, 0.8 g (7.84 mmol) of TMC and 0.16
g (0.83 mmol) of CDC were dissolved in 8 mL of dichloromethane and
placed to a seal reactor, and then 0.2 g (0.04 mmol) of
HO-PEG-OH10000 and 1 mL of catalyst solution of
bi(bistrimethylsilyl)amine zinc in dichloromethane (0.2 mol/L),
were added, then reacted in oil bath at 40.degree. C. for 2 days,
and subsequent processing steps were the same as those in Example
2, to obtain a triblock polymer
P(CDC3.8k-co-TMC18.8k)-PEG10k-P(CDC3.8k-co-TMC18.8k). The .sup.1H
NMR characteristic peak was the same as Example 2. The nuclear
magnetic resonance was used to calculate the following: m=227,
x=20, and y=184. Molecular weight measured by GPC: 92.3 kDa,
molecular weight distribution: 1.46.
##STR00013##
Example 10
Synthesis of a Diblock Polymer
NHS-PEG7.5k-P(CDC3.8k-co-LA13.8k)
[0048] Under nitrogen atmosphere, 0.1 g (0.52 mmol) of CDC and 0.4
g (2.8 mmol) of LA were dissolved in 3 mL of dichloromethane, and
placed to a seal reactor, and then 0.013 mmol of NHS-PEG 7500 and 1
mL of catalyst solution of (bis-trimethylsilyl)amine zinc in
dichloromethane (0.1 mol/L) were added. Then the reactor was sealed
and transferred out of the glove box. The mixture reacted in oil
bath at 40.degree. C. for 2 days. Subsequent processing steps were
the same as those in Example 2 to obtain
NHS-PEG7.5k-P(CDC4.8k-co-LA19.0k). .sup.1H NMR (400 MHz,
CDCl.sub.3): 1.59 (s, --COCH(CH.sub.3)O--), 3.08 (s, --CCH.sub.2),
3.30 (m, --OCH.sub.3), 3.65 (t, --OCH.sub.2CH.sub.2O--), 4.31 (m,
--CCH.sub.2), 5.07 (s, --COCH(CH.sub.3)O--) and 2.3 (s, NHS). The
nuclear magnetic resonance was used to calculate the following:
k=170, x=20, and y=96. Molecular weight measured by GPC: 42.3 kDa,
molecular weight distribution: 1.45.
##STR00014##
[0049] A variety of polymer containing dithiocarbonate monomers can
be prepared by the similar preparation methods described above. The
proportion and characterization of the raw materials are shown in
Table 1.
TABLE-US-00001 TABLE 1 Preparation condition for each polymer, and
nuclear magnetic resonance and GPC characterization results of the
product Molecular amount for repeated weight preparation (mmol)
unit (nuclear (kg/mol) PEG TMC, magnatic) Nuclear PDI Polymer
hydroxyl CDC LA, CL K or m x y magnatic GPC GPC PEG5k-P
(CDC4.9k-co- 0.02 0.52 3.85 114 26 186 28.9 34.5 1.48 TMC19k)
Mal-PEG6k-P (CDC4.8k- 0.017 0.52 3.85 136 25 188 29.9 38.6 1.42
co-TMC19.2k) NHS-PEG6.5k-P (CDC4.6k- 0.015 0.52 3.85 145 24 182
29.7 37.6 1.38 co-TMC18.6k) PEG5k-P (CDC5.8k-co- 0.02 0.52 4.90 114
30 226 33.8 45.6 1.53 TMC23k) NHS-PEG6.5k-P (CDC6k- 0.017 0.52 4.12
148 31 222 35.1 51.3 1.43 co-TMC22.6k) PEG7.5k-P (CDC5.8k-co- 0.01
0.31 1.93 170 30 125 33.3 54.5 1.36 TMC20.0k) PEG6.5k-P
(CDC5.8k-co- 0.015 0.57 3.47 148 30 200 36.6 42.4 1.43 LA28.3k)
PEG5k-P (CDC3.7k-co- 0.02 0.42 2.08 114 20 125 23.3 24.3 1.32
LA19.8k) Mal-PEG6k-P (CDC3.6k- 0.025 0.52 5.56 136 20 129 28.6 32.5
1.44 co-LA18.6k) P (CDC3.8k-co-TMC18.8k)- 0.01 0.21 1.96 272 20 184
57.2 84.5 1.53 PEG12k-P (CDC3.8k- co-TMC18.8k) P
(CDC3.8k-co-LA18.8k)- 0.05 0.52 2.08 227 20 131 55.2 92.3 1.46
PEG10k-P (CDC3.8k- co-LA18.8k) NHS-PEG7.5k-P (CDC3.8k- 0.013 0.52
2.80 170 20 131 24.1 42.3 1.45 co-LA18.8k) P (CDC3.8k-co-TMC18.8k)-
0.04 0.83 7.84 340 20 184 70.2 121.9 1.61 PEG15k-P (CDC3.8k-
co-TMC18.8k) AA-PEG5k-P (CDC3.8k- 0.02 0.42 3.92 114 20 189 28.1
34.6 1.43 co-TMC19.3k) PEG5k-P (CDC5.8k-co- 0.02 0.52 3.92 114 30
183 29.5 36.8 1.51 TMC18.7k) PEG5k-P (CDC5.7k-co- 0.02 0.52 2.08
114 30 131 29.5 40.9 1.42 LA18.8k)
Example 11
Synthesis of a Targeted Polymer
Transferrin-PEG7.5k-P(CDC3.8k-co-LA13.8k)
[0050] Transferrin-conjugated polymer is synthesized by two steps.
A first step is to prepare Mal-PEG7.5k-P(CDC3.8k-co-LA13.8k) as
described in Example 8; and a second step is to bond it with
transferrin via Michael reaction. The above described polymer
Mal-PEG7.5k-P(CDC3.8k-co-LA13.8K) was firstly dissolved in DMF,
into which 2 times molar amount of transferrin was added to carry
out reaction at 30.degree. C. for two days, and then the reaction
products were dialyzed, and lyophilized to obtain
transferrin-PEG6.5k-P(CDC3.8k-co-LA13.8k). The transferrin grafting
rate was calculated as 95% by the nuclear magnetic resonance and
BCA protein kit test.
Example 12
Synthesis of a Targeted Polymer
Herceptin-PEG6k-P(CDC3.6k-co-LA18.6k)
[0051] A human epidermal growth factor antibody
Herceptin-conjugated polymer is synthesized by three steps. The
first step is to prepare Mal-PEG6k-P(CDC3.6k-co-LA18.6k) as in the
Example 8, the second step is to convert the terminal group to an
amino group by reacting Mal-PEG6k-P(CDC3.6k-co-LA18.6k) with
cysteamine through Michael addition reaction; and the third step is
to bond a carboxyl of FA (folic acid) to the amino group by
amidation reaction. In detail, the polymer obtained in the previous
step is firstly dissolved in 0.5 ml of DMF, 2 ml of boric acid
buffer solution (pH 8.0) was added and then 2 times molar amount of
Herceptin was added to carry out reaction at 30.degree. C. for two
days, and then the reaction products were dialyzed and lyophilized
to obtain the Herceptin-PEG6k-P(CDC3.6k-co-LA18.6k). The
transferrin grafting rate was calculated as 96% by the nuclear
magnetic resonance and BCA protein kit test.
Example 13
Synthesis of a Targeted Polymer FA-PEG6.5k-P(CDC6k-co-TMC22.6k)
[0052] Folic acid (FA) coupled polymer is synthesized by two steps.
The first step is to prepare NHS-PEG6.5k-P(CDC6k-co-TMC22.6k) as in
the Example 4. The second step is to bond the amino group of FA to
NHS-PEG6.5k-P(CDC6k-co-TMC22.6k) by amidation reaction. In detail,
the above mentioned polymer is firstly dissolved in DMF, into which
2 times molar amount of FA was added to carry out reaction at
30.degree. C. for two days, and then the reaction products were
dialyzed to remove free FA and lyophilized to obtain
FA-PEG6.5k-P(CDC6k-co-TMC22.6k). The transferrin grafting rate was
calculated as 88% by the nuclear magnetic resonance.
Example 14
Synthesis of a Targeted Polymer
GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)
[0053] Cyclic polypeptide YHWYGYTPQNVI (GE11) coupled polymer is
prepared by two steps. The first step is to prepare
NHS-PEG6.5k-P(CDC6k-co-TMC22.6k) as in the Example 4. The second
step is to bond amino group of GE11 to the above product by
amidation reaction. In detail, the above mentioned polymer is
firstly dissolved in DMF, into which 2 times molar amount of GE11
was added to carry out reaction at 30.degree. C. for two days, and
then the reaction products were dialyzed to remove free GE11, and
lyophilized to obtain GE11-PEG6.5k-P(CDC6k-co-TMC22.6k). The GE11
grafting rate was calculated as 96% by the nuclear magnetic
resonance and BCA protein kit test.
[0054] According to the methods of Examples 11 to 13, the polymer
containing dithiocarbonate monomer in Examples 2 to 10 and Table 1
is bonded with a targeting molecule to prepare an ovarian cancer
specifically targeted degradable amphiphilic polymer.
Example 15
Preparation of Self-Crosslinked Polymeric Vesicles
[0055] Polymeric vesicles were prepared by solvent displacement 100
.mu.L of PEG5k-P(CDC5.8k-co-TMC23k) in DMF solution (10 mg/mL) was
added dropwise to 900 .mu.L of phosphate buffer solution (PB, 10
mM, pH 7.4), the mixed solutions were placed in a shaker at
37.degree. C. (200 rpm) overnight for self-crosslinked, and then
dialyzed overnight in a dialysis bag (MWCO 7000) with five times
replacement of media PB. FIG. 1 is a graph illustrating the
particle size distribution (A) and electron transmission microscopy
(B) of the self-crosslinked polymeric vesicle
PEG5k-P(CDC5.8k-co-TMC23k), a stability test (C) and a reduction
response test (D) of the cross-linked vesicles. The size of the
self-crosslinked polymeric vesicles obtained was determined by
dynamic light scattering particle size analyzer (DLS), and the
formed nano-vesicles have a particle size of 130 nm, and a very
narrow particle size distribution, as shown in FIG. 1A. It can be
found from FIG. B that, TEM shows that the nanoparticle has a
hollow vesicle structure, and the particle size and distribution
remains unchanged when the self-crosslinked polymeric vesicles were
highly diluted in the presence of fetal bovine serum (FIG. 1C).
However, in the case of simulation of tumor cell reduction
environment, the self-crosslinked polymeric vesicles released and
de-crosslinked rapidly (FIG. 1D). Thus, the obtained vesicles can
be self-crosslinked and have a property of reduction-sensitive
decrosslinking. Using the same preparation method,
PEG5k-P(CDC4.9k-co-TMC19k) can form self-crosslinked nanovesicles
with a particle size of 100 nm and a particle size distribution of
0.1.
Example 16
Preparation of Self-Crosslinked Polymeric Vesicles by Dialysis and
Thin-Film Hydration Methods
[0056] Self-crosslinked polymeric vesicles were prepared by
dialysis method. 100 .mu.L of PEG5k-P(CDC5.8k-co-TMC23k) in DMF (10
mg/mL) was placed in a dialysis bag (MWCO 7000) and placed in a
shaker at 37.degree. C. (200 rpm) overnight for crosslinking in
presence of PB, and then dialyzed for 24 hours in PB for five times
replacement of media PB. The cross-linked vesicles have a particle
size around 60 nm and a particle size distribution of 0.08 measured
by DLS.
[0057] Self-crosslinked polymeric vesicles were prepared by film
hydration method. 2 mg of PEG5k-P(CDC5.8k-co-TMC23k) was dissolved
in 0.5 mL of organic solvent with low boiling point, such as
dichloromethane or acetonitrile, in a 25 ml sharp-bottomed flask,
and rotary evaporated to form a film at the bottom. Then the flask
was evacuated for 24 hours under a vacuum of 0.1 mBar. 2 mL of PB
(10 mM, pH 7.4) was added and the solution was stirred, then a film
was peeled off and pulverized at 37.degree. C., and then sonicated
for 20 minutes (200 rpm), and continuously stirred for 24 hours.
The obtained vesicles were self-crosslinked. The size of the
self-crosslinked polymeric vesicles measured by DLS was about 160
nm and the particle size distribution was 0.24.
Example 17
Preparation of Self-Crosslinked Polymeric Vesicles GE11-CLPs Based
on PEG5k-P(CDC5.8k-co-TMC23k) and
GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)
[0058] The targeting polymer GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)
obtained in Example 14 and the PEG5k-P(CDC5.8k-co-TMC23k) obtained
in Example 2 were dissolved in DMF. The self-crosslinked polymeric
vesicle was prepared by a solvent displacement method as in Example
15. The PEG molecular weight of the targeting polymer is larger
than the non-targeting PEG, which ensures that the targeting
molecule has a better expenditure surface. The two kinds of
polymers can be mixed in different ratios to prepare
self-crosslinked polymeric vesicles GE11-CLPs with different
targeting molecular content on the surface. The targeting polymer
GE11-PEG6.5k-P(CDC6k-co-TMC22.6k) was used in an amount of 10 wt. %
to 30 wt. %. The self-crosslinked polymeric vesicles have a
particle size of about 85-130 nm and a particle size distribution
of 0.01-0.20 determined by DLS.
Example 18
Preparation of Self-Crosslinked Polymeric Vesicles FA-CLPs Based on
FA-PEG6.5k-P(CDC6k-co-TMC22.6k) and PEG5k-P(CDC5.8k-co-TMC23k)
[0059] A solution of 1.6 mg of the PEG5k-P(CDC5.8k-co-TMC23k)
prepared in Example 2 dissolved in DMF (10 mg/mL) and 0.4 mg of
FA-PEG6.5k-P(CDC6k-co-TMC22.6k) obtained in Example 13 dissolved in
0.5 mL of an organic solvent with low boiling point, such as
dichloromethane or acetonitrile were used to prepare the
self-crosslinked polymeric vesicle through the film hydration
method as in Example 17. The self-crosslinked polymeric vesicles
have a particle size of about 88 nm and a particle size
distribution of 0.08. The two kinds of polymers can be mixed in
different ratios to prepare self-crosslinked polymeric vesicles
FA-CLPs with different targeted molecular content on the surface.
The FA-PEG6.5k-P(CDC6k-co-TMC22.6k) was used in an amount of 5-30
wt. %. The self-crosslinked polymeric vesicles have a particle size
of about 85-130 nm and a particle size distribution of
0.01-0.20.
Example 19
Preparation of Self-Crosslinked Polymeric Vesicles Transferrin CLPs
With Surface Coupled to Transferrin
[0060] Mal-PEG6k-P(CDC3.6k-LA18.6k) prepared in Example 8 was mixed
with P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k), then 0.5 ml of 4M
boric acid buffer solution (pH 8.0) was added to adjust the pH of
the solution to 7.5-8.0, and transferrin was added in an amount of
1.5 times the molar amount of Mal. They bonded to each other
through Michael addition reaction, and reacted at 30 C. for two
days, and then the reaction products were dialyzed, and prepared
into vesicles transferrin-CLPs according to the dialysis method in
Example 16. The vesicles were measured by DLS to have a particle
size of 115 nm and a particle size distribution of 0.12. The graft
ratio of the polypeptides was calculated via nuclear magnetic and
BCA protein kits to be 94%. The targeting polymer and non-targeting
polymer were fed by mass ratio in order to prepare self-crosslinked
polymeric vesicles transferrin-CLPs from different targeting
molecules, wherein the vesicles transferrin-CLPs have a particle
size of about 85-130 nm, and a particle size distribution of
0.01-0.20.
[0061] A variety of self-crosslinked polymeric vesicles can be
prepared by similar preparation methods described above, wherein
the proportions and characterization of the raw materials are shown
in Table 2.
TABLE-US-00002 TABLE 2 Preparation and characterization of
self-crosslinked polymeric vesicles solvent filming- particle
displacement dialysis rehydroation size size Polymer method method
method (nm) distribution PEG5k-P(CDC3.9k-co-LA18.6k) 130 0.14
PEG5k-P(CDC5.8k-co-TMC23k) 102 0.11 FA/PEG5k-P(CDC5.8k-co-TMC23k)
88 0.08 GE11/PEG5k-P(CDC5.8k-co-TMC23k) 96 0.18
PEG5k-P(CDC4.9k-TMC14.1k) 67 0.12 PEG6.5k-P(CDC5.8k-LA28.3k) 158
0.20 PEG7.5k-P(CDC3.9k-TMC14.1k) 53 0.15 P(CDC3.9k-TMC18.8k)-PEG5k-
143 0.21 P(CDC3.9k-TMC18.8k) N.sub.3-PEG7k-P(CDC4.7k-PDSC12.6k) 100
0.13 AA-PEG5k-P(CDC3.9k-PDSC14.8k) 152 0.18
Ally-PEG6k-P(CDC3.9k-CL14.2k) 124 0.21
Example 20
Drug Loading and In Vitro Release of Self-Crosslinked Polymeric
Vesicles
[0062] PEG5k-P(CDC5.8k-co-TMC23k) self-crosslinked polymer vesicles
were prepared by solvent displacement method. DOX.HCl was loaded by
pH gradient method utilizing different pH inside and outside the
vesicles to have the hydrophilic DOX.HCl encapsulated. 100 .mu.L of
PEG5k-P(CDC5.8-co-TMC23k) in DMF solution (10 mg/mL) was added
dropwise to 900 .mu.L sodium citrate/citrate acid buffer solution
(10 mM, pH 4.0), and placed in a shaker at 37.degree. C. (200 rmp)
for 5 hours, then 0.05 mL of PB (4 M, pH 8.1) was added to
establish aa pH gradient, then the DOX.HCl was immediately added
and placed in the shaker for 5-10 hours to allow the drug to enter
the vesicles while the vesicles were self-crosslinked. Finally,
they were dialyzed overnight in a dialysis bat (MWCO 7000), and the
PB (10 mM, pH 7.4) for dialysis medium was changed five times. The
self-crosslinked polymeric vesicles carrying different proportions
of the drug (10%-30%) have a particle size of 108-128 nm and a
particle size distribution of 0.10-0.14. Encapsulation efficiency
of DOX.HCl measured by Fluorescence spectrometer was 68% to 85%.
The in vitro release experiments of DOX.HCl was performed by
shaking (200 rpm) at a constant temperature shaker of 37.degree. C.
with three parallel samples in each group. In the first group,
DOX.HCl-loaded self-crosslinked polymeric vesicles in PB (10 mM, pH
7.4) added with 10 mM GSH simulating intracellular reducing
environment: the second group, DOX.HCl-loaded self-crosslinked
polymeric vesicles in PB (10 mM, pH 7.4). The concentration of
drug-carrying self-crosslinked polymeric vesicles was 30 mg/L,
wherein 0.6 mL was placed in a dialysis bag (MWCO: 12,000), and
each tube was filled with 25 mL of the corresponding dialysis
solvent. 5.0 mL dialysis solvent outside the dialysis bag was taken
out at the scheduled time interval for test and 5.0 mL of the
corresponding media was supplemented to the tube. The concentration
of the drug in the solution was measured by a fluorometer. FIG. 2
shows the relationship between the cumulative release of DOX.HCl in
respective of time. It can be found from the figure that the
release of DOX.HCl in the sample added with GSH is significantly
faster than that in the samples without GSH, indicating that the
drug-carrying self-crosslinked polymeric vesicles are capable of
releasing drug effectively in the presence of 10 mM GSH.
[0063] The DOX.HCl-loaded self-crosslinked polymeric vesicle
PEG5k-P(CDC4.9k-co-TMC19k) was prepared by the same method as
above. The self-crosslinked polymeric vesicles comprising different
proportions of the drug (10% to 30%) have a particle size of
100-125 nm and a particle size distribution of 0.10-0.14.
Encapsulation efficiency of DOX.HCl measured by Fluorescence
spectrometer was 60%-80%.
[0064] Self-crosslinked polymeric vesicles
Ally-PEG6k-P(CDC2.9k-CL14.2k) were prepared by solvent
displacement. 10 .mu.L of paclitaxel PTX in DMF (10 mg/mL) and 90
.mu.L of Ally-PEG6k-P(CDC2.9k-CL14.2k) in DMF (10 mg/mL) were
mixed, then added dropwise to 900 .mu.L of phosphate buffer (10 mM,
pH 7.4, PB), and placed in a shaker at 37.degree. C. (200 rpm) for
self-crosslinked overnight, then placed in a dialysis bag (MWCO
7000) for dialysis overnight, during which water was refreshed for
five times. The dialysis medium is PB (10 mM, pH 7.4). The PTX is
in an amount of 0-20 wt. %, and the obtained self-crosslinked
polymeric vesicles have a particle size of 130-170 nm and a
particle size distribution of 0.1-0.2. The vesicle structure
measured by TEM has decrosslinking properties sensitive to
reduction. The encapsulation efficiency of PTX is 50%-70%. The in
vitro release experiment was designed as above, and the release of
hydrophobic drugs in the sample with GSH was significantly faster
than that in the sample without GSH.
[0065] Drug-loaded self-crosslinked polymeric vesicles FA-CLPs
based on PEG6.5k-P(CDC6k-co-TMC22.6k) were prepared by
filming-rehydration method, and DOX.HCl was loaded by pH gradient
method. 1.6 mg of PEG5k-P(CDC5.8k-co-TMC23k) and 0.4 mg of
FA-PEG6.5k-P(CDC6k-co-TMC22.6k) were dissolved in 0.5 mL of low
boiling organic solvent such as dichloromethane or acetonitrile,
and rotary evaporated in a 25 ml sharp-bottomed flask to form a
film at the bottom. Then the flask was evacuated under a vacuum of
0.1 mBar for 24 hours. 2 mL of sodium citrate/citrate acid buffer
solution (10 mM, pH 4.0) was added, then the film was peeled off
the bottom at 37.degree. C. and stirred to pieces. Ultrasonic
irradiation (200 rpm) was carried out for 20 minutes, stirring for
24 hours, then the vesicles were self-crosslinked. The crosslinking
vesicles were measured by DLS to have a particle size of 90 nm and
a particle size distribution of 0.10. 0.05 mL of PB (4M, pH 8.1)
was added to the above solution of vesicles to establish a pH
gradient, followed by immediate addition of DOX.HCl, and placed in
a shaker for 5-10 hours. The above mixed solution was then placed
in a dialysis bag (MWCO 7000) and dialyzed with PB overnight,
during which water was replaced for five times. After loaded with
different proportions (10%-30%) of drugs, the vesicles have a
particle size of 112-121 nm, and a particle size distribution of
0.10-0.15. The encapsulation efficiency of DOX.HCl is 61%-77%. The
in vitro release experiment was designed as above, and the release
of drugs in the sample with 10 mM GSH was significantly faster than
that in the sample without GSH.
[0066] The drug-carrying self-crosslinked polymeric vesicle
GE11-CLPs based on PEG6.5k-P(CDC6k-co-TMC22.6k) was prepared by
dialysis method, and doxorubicin hydrochloride (Epi.HCl) was loaded
by pH gradient method. 80 .mu.L of PEG5k-P(CDC5.8k-co-TMC23k) in
DMF (10 mg/mL) and 20 .mu.L of GE11-PEG6.5k-P(CDC6k-co-TMC22.6k) in
DMF (10 mg/mL) were mixed uniformly, then directly placed into a
dialysis bag (MWCO 7000), placed in a sodium citrate/citrate buffer
solution (10 mM, pH 4.0), placed in a 37.degree. C. shaker for 4
hours to carry out self-crosslinked. After dialysis for the same
medium for 12 hours, change the liquid five times. The cross-linked
vesicles have a particle size of 96 nm with a particle size
distribution of 0.18 measured by DLS. 0.05 mL of PB (4M, pH 8.5)
was added to the above vesicle solution to establish a pH gradient,
followed by immediately adding Epi.HCl and placing in a shaker for
5-10 hours. It was then placed in a dialysis bag (MWCO 7000) and
dialyzed against PB overnight for five times. Loaded in different
proportions (10%-30%), particle size 98-118 nm, particle size
distribution 0.10-0.15, DOX.HCl package efficiency 64%-79%. The
design of Epi.HCl in vitro release was the same as above, as shown
in FIG. 3. After adding 10 mM GSH, the drug was effectively
released at a faster rate than the sample without GSH.
[0067] Similar preparation methods as described above can be used
to study the drug loading capacity and encapsulation efficiency
effects of various self-crosslinked polymeric vesicles and targeted
self-crosslinked polymeric vesicles to a variety of hydrophilic
anti-cancer small molecule drugs and genes, such as doxorubicin
hydrochloride (DOX.HCl), Epirubicin hydrochloride (Epi.HCl),
irinotecan hydrochloride (CPT.HCl) and mitoxantrone hydrochloride
(MTO.HCl) and the hydrophobic anticancer drugs paclitaxel,
docetaxel and olaparib. The loading amount and package efficiency
are shown in Table 3.
TABLE-US-00003 TABLE 3 Drug loading capacity for hydrophilic drugs
and encapsulation efficiency of self- crosslinked polymeric
vesicles and targeting self-crosslinked polymeric vesicles Feed
Drug encapsulation Particle particle ratio loading rate size size
Polymer/drug (wt. %) (wt. %) (%) (nm) distribution
PEG5k-P(CDC3.9k-co-LA14.6k)/DOX.cndot.HCl 0 -- -- 123 0.05 10 6.5
69.7 134 0.14 20 12.3 69.9 142 0.18 30 20.9 88.3 153 0.18
PEG5k-P(CDC5.8k-co-TMC23k)/DOX.cndot.HCl 0 -- -- 102 0.11 10 7.1
76.5 105 0.11 20 11.9 67.6 108 0.12 30 15.9 62.9 124 0.15
FA20/PEG5k-P(CDC5.8k-co-TMC23k)/DOX.cndot.HCl 0 -- -- 88 0.08 10
7.2 77.2 112 0.10 20 11.8 66.8 116 0.13 30 15.4 60.5 121 0.15
GE1120/PEG5k-P(CDC5.8k-co-TMC23k)/DOX.cndot.HCl 0 -- -- 96 0.18 10
7.3 79.1 98 0.10 15 10.3 76.8 105 0.13 20 11.4 64.3 118 0.15
transferrin-PEG6.5k-P(CDC3.8k-co-LA13.8k)/DOX.cndot.HCl 0 -- -- 134
0.09 20 13.9 80.5 168 0.21 PEG5k-P(CDC4.9k-TMC14.1k)/DOX.cndot.HCl
0 -- -- 40 0.12 20 11.9 67.3 52 0.18
PEG7.5k-P(CDC4.9k-TMC14.1k)/DOX.cndot.HCl 0 -- -- 57 0.19 20 11.5
64.8 63 0.17
P(CDC3.9k-TMC18.8k)-PEG5k-P(CDC3.9.k-TMC18.8k)/Epi.cndot.HCl 0 --
-- 143 0.21 20 12.6 71.8 172 0.26
FA/PEG5k-P(CDC5.8k-co-TMC23k)/CPT.cndot.HCl 20 12.6 72.1 138 0.18
GE11/PEG5k-P(CDC5.8k-co-TMC23k)/MTO.cndot.HCl 20 6.7 35.8 108 0.10
transferrin/P(CDC3.9k-LA18.8k)-PEG4k- 20 11.9 67.5 121 0.06
P(CDC3.9k-LA18.8k)/CPT.cndot.HCl Ally-PEG6k-P(CDC4.9k-CL14.2k)/PTX
20 13.5 78.1 135 0.17
Example 21
Test of the Toxicity of the the Empty Self-Crosslinked Polymeric
Vesicles to SKOV3 Cells by MTT Method
[0068] The cytotoxicity of the empty polymeric vesicles to SKOV3
human ovarian cancer cells was tested by MTT method. SKOV3 cells
were seeded in 96-well plates at 5.times.10.sup.4 cells/mL, 100
.mu.L per well, and the cells were cultured for 24 hours until 70%
of cells were adhered to wall. Then, vesicle samples (such as
non-loading self-crosslinked polymeric vesicles of Example 15 and
Example 19) with different concentrations (0.5, 1.0 mg/mL) were
added to each well of the experimental group. Cells of control well
and medium of control wells (4 wells repeated) were further
arranged. After 24 hours of culture, 10 .mu.L of MTT (5.0 mg/mL)
was added to each well for further incubation for 4 hours, then 150
.mu.L of DMSO was added to each well to dissolve the crystals
produced, and the absorbance value (A) was measured at 492 nm using
a microplate reader to calculate cell viability with the medium of
control well as zero. FIG. 4 is a graph illustrating the
cytotoxicity of self-crosslinked polymeric vesicles. It can be
found that when the concentration of self-crosslinked polymeric
vesicles increased from 0.5 to 1.0 mg/mL, the survival rate of
SKOV3 was still higher than 92% indicating that the
self-crosslinked polymeric vesicles of the present invention have
good biocompatibility.
Example 22
Test of Toxicity of Drug-Loaded Self-Crosslinked Polymeric Vesicles
to SKOV3 Ovarian Cancer Cells by MTT Method
[0069] The culture of cells was the same as that in Example 21
except that: DOX.HCl-loaded self-crosslinked polymeric vesicles
PEG5k-P(CDC5.8k-co-TMC23k), DOX.HCl-loaded self-crosslinked
polymeric vesicles GE11-CLPs composed of PEG5k-P(CDC5.8k-co-TMC23k)
and GE11-PEG6.5k-P(CDC6k-co-TMC22.6k) (wherein GE11 were in an
amount of 10%, 20%, 30%, respectively) were added to each
corresponding well of the experimental group. The concentration of
DOX.HCl were 0.01, 0.1, 0.5, 1, 5, 10, 20, 40 and 80 .mu.g/mL; and
the targeting molecules were in an amount of 10%, 20% and 30%. The
adriamycin liposomes Libod group was set as a control group. After
4 hours of co-cultivation, the samples were aspirated and cultured
in fresh medium fur a further cultivation for 44 h. The addition
and process of MTT and the measuring of the absorbance were the
same as in Example 21. FIG. 5 and FIG. 6 show the toxicity of
drug-loaded self-crosslinked polymeric vesicles GE11-CLPs GE11 to
SKOV3 cells. It can be found that 20% DOX.HCl-loaded
self-crosslinked polymeric vesicles GE11-CLPs GE11 loading have a
semi-lethal concentration (IC50) of 2.01 .mu.g/mL to SKOV3 cells,
which is much lower than that of self-crosslinked polymeric
vesicles PEG5k-P(CDC5.8k-co-TMC23k), and also lower than that of
adriamycin liposomes Libod (14.23 .mu.g/mL), indicating that the
drug-carrying self-crosslinked polymeric vesicles of the present
invention can effectively target ovarian cancer cells to release
drugs in cells, and finally kill cancer cells.
Example 23
Test of Toxicity of Drug-Loaded Self-Crosslinked Polymeric Vesicles
to A2780 Cells by MTT Method
[0070] The culture of the cells was the same as in Example 21,
except adding the self-crosslinked polymeric vesicles with
different transferrin contents and different doses of drugs for the
wells of the experimental group. For example, CPT.HCl, and
self-crosslinked polymeric vesicles transferrin-CLPs prepared from
Mal-PEG6k-P(CDC3.6k-LA18.6k) and
P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k) (Example 19) were added
to corresponding wells, wherein the concentration of CPT.HCl is
0.01, 0.1, 0.5, 1, 5, 10, 20 and 40 .mu.g/mL; the targeting
molecular is in an amount of 10%, 20% and 30%; and the
drug-carrying crosslinking polymeric vesicles
P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k) and a CPT.HCl group
were set as a control group. After 4 hours of co-cultivation, the
samples were aspirated and cultured in fresh medium for a further
44 h. Subsequent addition, process and measurement for absorbance
of MTT were the same as in Example 21. The results showed that the
IC.sub.50 of the drug-loaded self-crosslinked polymeric vesicles
P(CDC3.8k-LA18.8k)-PEG4k-P(CDC3.8k-LA18.8k) to A2780 cells was 4.15
.mu.g/mL, and the IC.sub.50 of 30% transferrin-CLPs carrying
CPT.HCl to A2780 cells is 2.07 .mu.g/mL, which was lower than that
of the drug CPT.HCl (4.11 .mu.g/mL).
[0071] Therefore, the drug-loaded self-crosslinked polymeric
vesicle of the invention can effectively target ovarian cancer
cells to release drugs in cells, and finally kill cancer cells.
Especially the binding of targeting molecules greatly enhances the
specificity to ovarian cancer cells and significantly increases the
lethality of drugs against ovarian cancer cells.
[0072] The toxicity of various drug-loaded self-crosslinked
polymeric vesicles on ovarian cancer cells was studied by similar
methods described above. The drugs are a hydrophilic anti-cancer
small molecule drug and the gene drug, such as doxorubicin
hydrochloride (DOX.HCl), Epirubicin hydrochloride (Epi.HCl),
irinotecan hydrochloride (CPT.HCl) and mitoxantrone hydrochloride
(MTO.HCl) and the hydrophobic anticancer drugs, such as paclitaxel,
docetaxel and olaparib, and the results are shown in Table 4.
Example 24
Endocytosis of Drug-Loaded Self-Crosslinked Polymeric Vesicles
(CLPs and GE11-CLPs)
[0073] Flow cytometry was used to test the endocytosis of
drug-carrying vesicles using SKOV3 human ovarian cancer cells.
SKOV3 cells were seeded in 6-well plates at 2.times.10.sup.5
cells/mL, 900 .mu.L per well, and the cells were cultured for 24
hours until 70% cells are adherent to wall. Then, the drug-carrying
vesicles samples CLPs and GE11-CLPs were added to each well of the
experimental group. Cells of control well and medium of control
wells (2 wells repeated) were further arranged. After 4 hours of
culture, each well was trypsinized for 5 minutes, and washed three
times with physiological saline. The fluorescence absorption
intensity was measured at 488 nm by flow cytometry, and the
endocytosis amount was calculated by using the saline group as a
control. FIG. 7 shows the results of cell uptake of drug-loaded
cross-linked polymeric vesicles. It can be found that the uptake of
cells by targeted drug-loaded self-crosslinked polymeric vesicles
is higher than that of non-targeting drug-carrying self-crosslinked
polymeric vesicles and adriamycin liposomes Libod, indicating that
the targeted self-crosslinked polymeric vesicles can be actively
taken up via endocytosis by ovarian cancer cells.
Example 25
Blood Circulation of Drug-Loaded Self-Crosslinked Polymeric
Vesicles (CLPs and FA-CLPs)
[0074] All animal experiments were conducted in accordance with the
regulations of the Animal Experimental Center of Suzhou University.
Balb/C nude mice 18-20 grams and 4-6 weeks old were used in the
experiments. The vesicles were prepared by mixing
FA-PEG6.5k-P(CDC6k-co-TMC22.6k) and PEG5k-P(CDC5.8k-co-TMC23k) in
different proportions, designated as FA-CLPs. When the proportion
of FA in the polymeric vesicles was 20%, the particle size is 100
nm, the particle size distribution is 0.10, and the polymeric
vesicles was designated as FA20CLPs, the drug was DOX.HCl.
DOX.HCl-loaded CLPs vesicles, FA-CLPs vesicles, and DOX.HCl were
injected into mice via tail vein (DOX dose of 10 mg/kg). 10 .mu.L
blood was taken after 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours
respectively. The blood was accurately weighed by the differential
method, then 100 .mu.L of 1% Triton and 500 .mu.L of extraction
liquid (DMF comprising 20 mM DTT and 1 M HCl) were added; then
centrifugation (20,000 rpm, 20 minutes) was performed to obtain a
supernatant. The amount of DOX.HCl at each time point was measured
by fluorescence. It can be found from the calculation that the
elimination half-lives of drug-loaded self-crosslinked polymeric
vesicles FA-CLPs and drug-loaded self-crosslinked polymeric
vesicles CLPs in mice were 4.23 and 4.16 hours respectively, while
DOX.HCl has an elimination half-life of 0.27 hours. Therefore the
self-crosslinked polymeric vesicles disclosed herein are stable in
mice and have a long cycle time. The operation and calculation
methods of the blood circulation experiments for other
drug-carrying self-crosslinked polymeric vesicles were the same
with the above description, and the results are shown in Table
4.
Example 26
Blood Circulation of Drug-Loaded Self-Crosslinked Polymeric Vesicle
CLPs and GE11-CLPs
[0075] As in Example 25, self-crosslinked polymeric vesicles
GE11-CLPs were prepared by mixing GE11-PEG6.5k-P(CDC6k-co-TMC22.6k)
and PEG5k-P(CDC5.8k-co-TMC23k). Self-crosslinked polymeric vesicles
GE11-CLPs and self-crosslinked polymeric vesicles CLPs were loaded
with DOX.HCl, and then injected into Balb/C nude mice via tail vein
to study blood circulation thereof, wherein DOX.HCl and Libod
DOX-LPs were used for control group. As shown in FIG. 10, the
GE11-CLPs and CLPs vesicles remained 5.0 ID%/g after 48 hours. it
can be calculated that the elimination half-life of
self-crosslinked polymeric vesicles GE11-CLPs and self-crosslinked
polymeric vesicles CLPs in mice are 4.99 and 4.79 hours
respectively, therefore they are stable in mice and have a long
cycle time. The results are shown in Table 4.
Example 27
In Vivo Imaging of Self-Crosslinked Polymeric Vesicles FA-CLPs in
SKOV3 Ovarian Cancer Mice
[0076] In vivo imaging experiments were performed on Balb/C nude
mice of 4-6 weeks old weighing about 18-20 g. The mice were
subcutaneously injected with 5.times.10.sup.6 SKOV3 human ovarian
cancer cells. The experiments were started after about 3-4 weeks
when the tumor grows to 100.about.200 mm.sup.3. Self-crosslinked
polymeric vesicles FA20-CLPs prepared by mixing
FA-PEG6.5k-P(CDC6k-co-TMC22.6k) with PEG5k-P(CDC5.8k-co-TMC23k) in
a ratio of 1:5 and self-crosslinked polymeric vesicles CLPs are
exemplified. The FA20-CLPs labeled by fluorescent substance cy-7
and the non-targeted CLPs were injected into the mice via tail
vein, and then the vesicles were tracked at different time points
1, 2, 4, 6, 8, 12, 24, 48 hours with living small animal optical
imaging system. The experimental results show that FA20-CLPs
accumulate rapidly at the tumor site, and the fluorescence is still
strong after 48 hours. These results indicate that FA20-CLPs can
actively target ovarian cancer tumor sites and be enriched there,
showing strong specificity for ovarian cancer cells. The operation
and calculation methods of the in vivo imaging experiments for
other self-crosslinked polymeric vesicles were the same with the
above description, and the results are shown in Table 4.
[0077] Epi.HCl-loaded cy-7-labeled CLPs and GE11-CLPs were prepared
to perform the in vivo imaging experiments, wherein the tumor
inoculation and tail vein administration were the same as the above
description. The both vesicles were found to accumulate rapidly at
ovarian tumor sites. LPs disappeared in 4-6 hour, but the
fluorescence of the tumor site with GE11-CLPs remained strong after
48 hours, indicating that GE11-CLPs can actively target the ovarian
tumor site and be enriched there.
Example 28
In Vivo Imaging Experiments of Drug-Loaded Self-Crosslinked
Polymeric Vesicle CLPs and Transferrin-CLPs in A2780 Ovarian Cancer
Mice
[0078] In vivo imaging experiments were performed on Balb/C nude
mice of 4-6 weeks old weighing about 18-20 g. The mice were
subcutaneously injected 5.times.10.sup.6 A2780 human ovarian cancer
cells. The experiments were started after about 3.about.4 weeks
when the tumor grows to 100-200 mm.sup.3.
[0079] Targeting self-crosslinked polymeric vesicles
transferrin-CLPs prepared by mixing
ferferrin-PEG6.5k-P(CDC3.8k-co-LA13.8k) and
PEG5k-P(CDC3.7k-co-LA14.6k) and drug-loaded self-crosslinked
polymeric vesicles CLPs were labeled with cy-5 and loaded with the
hydrophobic drug docetaxel DTX. The same procedure as in Example 27
was used to study in vivo imaging. The experimental results show
that the DTX carrying-transferrin-CLPs can accumulate rapidly in
the tumor site, and the fluorescence of that tumor site was still
strong after 48 hours indicating that transferrin-CLPs can actively
target tumor sites and be enriched there, while drug-loaded CLPs
self-crosslinked polymeric vesicles metabolize quickly after
entering the tumor in 2 hours, and the fluorescence intensity is
low.
Example 29
Biodistribution of Drug-Loaded Self-Crosslinked Polymeric Vesicles
CLPs and FA-CLPs in SKOV3 Ovarian Cancer Mice
[0080] Preparation of sample FA20CLPs and inoculation of tumors and
tail vein administration in biodistribution experiments were the
same as in Example 27. FA20-CLPs and CLPs were injected into mice
(DOX.HCl, 10 mg/kg). The mice were sacrificed after 12 hours, and
the tumor, heart, liver, spleen, lung and kidney tissues were
obtained and washed and weighed, then 500 .mu.L 1% Triton was added
respectively, and the tissue was grounded by a homogenizer and then
extracted with 900 .mu.L of DMF (comprising 20 mM DTT, 1 M HCl).
After centrifugation (20,000 rpm for 20 minutes), a supernatant was
obtained and measured by fluorescence at each time point for the
amount of DOX.HCl. In FIG. 8, the horizontal axis represents the
tissue organ, and the vertical axis represents the amount of
DOX.HCl account for total DOX.HCl injection (ID%/g) per gram of
tumor or tissue. The amount of DOX.HCl accumulated in tumors 12
hours after injection of FA-CLPs, CLPs and DOX.HCl were 6.54, 2.53
and 1.02 ID%/g respectively. The amount of DOX.HCl for FA-CLPs were
3 and 6 times higher than that of CLPs and DOX.HCl, indicating
drug-loaded FA-CLPs accumulate more at the tumor site by active
targeting, and have significant specificity to ovarian cancer
cells, which is beneficial for killing ovarian cancer cells. The
results are shown in Table 4.
Example 30
Biodistribution of Drug-Loaded Self-Crosslinked Polymeric Vesicle
CLPs and GE11-CLPs in SKOV3 Ovarian Cancer Mice
[0081] Tumor inoculation, tail vein administration, and operations
for animals are the same as in Example 27. GE11-CLPs and CLPs
carrying DOX.HCl and liposomal doxorubicin Libod DOX-LPs were
injected into mice (DOX.HCl: 10 mg/kg) via tail vein. After 6
hours, the amount of DOX.HCl accumulated in the tumors for
GE11-CLPs, CLPs and DOX-LP were 8.63, 3.52 and 1.82 ID%/g,
respectively, and the amount of DOX.HCl for GE11-CLPs were 2 and 5
times higher than the latter two, indicating that the drug-loaded
GE11CLP accumulates more at the tumor site by active targeting
(FIG. 9).
Example 31
The Maximum Tolerated Dose (MTD) of Balb/C Mice to Drug-Loaded
Self-Crosslinked Polymer Vesicles GE11-CLPs
[0082] Balb/C nude mice of 4.about.6 weeks old, weighing about
18.about.20 g were used. Self-crosslinked polymeric vesicles
GE11-CLPs and doxorubicin liposomes Libod (including doxorubicin at
concentrations of 120 mg/kg, 140 mg/kg, 160 mg/kg, 180 mg/kg and
200 mg/kg,) were injected at a single-dose, and the concentration
of doxorubicin liposomes Libod was 20 mg/kg. There are five mice in
each group. The mental state of the mice was observed and the body
weight was measured for the last 10 days before executed. The
standard of the maximum tolerated dose was that the mice did not
die accidentally or the body weight of the mice did not decrease to
below 15% of the mice. It can be seen from the changes of body
weight and survival rate of the mice for each component in FIGS.
10A and 10B, the maximum tolerated dose of the drug-loaded targeted
self-crosslinked polymeric vesicles was 160 mg/kg, while the
maximum tolerated dose of doxorubicin liposomes Libod was 20 mg/kg.
Therefore, the mice have high tolerance to targeted drug-loaded
self-crosslinked polymeric vesicles which greatly improves the
therapeutic window.
Example 32
Antitumor Effect of Drug-Loaded Self-Crosslinked Polymeric Vesicles
GE11-CLPs and CLPs and Effects Thereof to Body Weight Change and
Survival Rate for Mice Bearing SKOV3 Subcutaneous Ovarian
Cancer
[0083] Balb/C nude mice of 4-6 weeks old, weighing about 18-20 g
were injected subcutaneously with 5.times.10.sup.6 SKOV3 human
ovarian cancer cells. The experiment was started after about two
weeks when the tumor grows to 30.about.50 mm.sup.3. DOX.HCl-loaded
self-crosslinked polymeric vesicles GE11-CLPs prepared by mixing
GE11-PEG6.5k-P(CDC6k-co-TMC22.6k) and PEG5k-P(CDC5.8k-co-TMC23k) at
1:5, the non-targeted CLPs, DOX-LPs, and PBS were injected via tail
vein. As shown in FIG. 11, tumors were significantly inhibited in
the GE11-CLPs treatment group at 18th day, while tumors in the
drug-carrying CLPs group increased, and the body weight of the mice
hardly changed. Although DOX-LPs also inhibited tumor growth, the
body weight of mice in the DOX-LPs group decreased by 18% at 12th
day, indicating serious toxic side effects thereof on mice. The
GE11-CLPs treatment group survived after 62 days, the DOX-LPs group
had all died at 42th day, and the PBS group also all died at 42th
day. Therefore, drug-loaded self-crosslinked polymeric vesicles can
effectively inhibit tumors, have no toxic side effects on mice, and
prolong the survival time of tumor-bearing mice.
Example 33
Antitumor Effect of Drug-Carrying Self-Crosslinked Polymeric
Vesicles GE11-CLPs at Single Dose and Effects Thereof to Body
Weight Change and Survival Rate for Mice Bearing SKOV3 Subcutaneous
Ovarian Cancer
[0084] The establishment of subcutaneous SKOV3 tumor model, tail
vein administration and data collection were the same as in Example
32. DOX.HCl-loaded GE11-CLPs, doxorubicin liposomes Libod DOX-LPs,
and PBS were injected via tail vein at a single dose, wherein the
DOX.HCl-loaded self-crosslinked polymeric vesicles GE11-CLPs had
doxorubicin concentrations of 20 mg/kg, 40 mg/kg and 60 mg/kg,
while the doxorubicin dose of DOX-LPs had doxorubicin
concentrations of 10 mg/kg and 15 mg/kg. As shown in FIG. 12, in
GE11-CLPs group with 60 mg/kg of doxorubicin, the tumors were
significantly inhibited at 18th day, while in GE11-CLPs groups with
20 mg/kg and 40 mg/kg of doxorubicin, the tumors grew, and the body
weight of the mice in all groups hardly changed. DOX-LPs with 10
mg/kg and 15 mg/kg of doxorubicin did not inhibit tumor growth at a
single dose. The GE11-CLPs treatment group survived after 49th day,
the DOX-LPs group had all died at 42th and 43th day, and the PBS
group had also all died at 34th day.
Example 34
Antitumor Effect of Drug-Loading Targeted Self-Crosslinked
Polymeric Vesicles Transferrin-CLPs and CLPs and Effects Thereof to
Body Weight Change and Survival Rate for Mice Bearing A2780
Subcutaneous Ovarian Cancer
[0085] The establishment of the subcutaneous A2780 tumor model, way
of tail vein administration and data collection were the same as in
Example 32. The experiment was started at a tumor size of 30-50
mm.sup.3. CPT.HCl-loaded targeted self-crosslinked polymeric
vesicles transferrin-CLPs prepared by mixing
transferrin-PEG6.5k-P(CDC3.8k-co-LA13.8k) and
PEG5k-P(CDC3.7k-co-LA14.6k) at 1:5, the non-targeted CLPs, free
CPT.HCl were injected via tail vein. DOX.HCl-carrying
self-crosslinked polymeric vesicle cRGD-CLPs prepared by mixing
cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k) and PEG5k-P(CDC4.9k-co-TMC19k) at
1:5 was set as a control group. The results showed that tumors were
significantly inhibited at 18th day when treated with
transferrin-CLPs, while tumors in the drug-carrying CLPs group
showed a small increase, and the body weight of the mice didn't
change. The weight of mice in the cRGD-CLPs group did not change,
but the tumor inhibition was significantly weaker than the former,
and the tumor size was 3 times that of the former, indicating that
cRGD had no obvious targeting property to ovarian cancer. Although
CPT.HCl also inhibited growth of tumor, the weight of mice in the
CPT.HCl group decreased by 18% at 10th day. Mice in the
transferrin-CLPs treatment group had all survived after 72 days,
while mice in the CPT.HCl group had all died at 28th day, and the
PBS group also had all died at 37th day.
Example 35
Antitumor Effect of Drug-Carrying Targeting Self-Crosslinked
Polymeric Vesicles GE11-CLPs and CLPs and Effects Thereof to Body
Weight Change and Survival Rate for Mice Bearing SKOV3 Orthotopic
Ovarian Cancer
[0086] DOX.HCl-carrying self-crosslinked polymeric vesicles
GE11-CLPs, the non-targeted CLPs, DOX-LPs, and PBS were injected
into mice bearing SKOV3 orthotopic ovarian cancer via tail vein. In
the GE11-CLPs treatment group, the bioluminescence intensity of the
tumor continued to decrease within 16 days, while the
bioluminescence intensity of the tumor in drug-loaded CLP group
increased to some extent, and the body weight of the mice hardly
changed. Although DOX-LPs can also inhibit tumor growth, the body
weight of mice in DOX-LPs group decreased by 21% at 4th day. The
mice in GE11-CLPs treatment group had all survived after 45 days,
the mice in DOX-LPs group had all died at 32th day, and the mice in
PBS group had also all died at 23th day. Therefore, the drug-loaded
self-crosslinked polymeric vesicles GE11-CLPs bonded to targeted
molecules can effectively inhibit the growth of orthotopic ovarian
cancer, have no toxic side effects, and can prolong the survival
time of tumor-bearing mice.
[0087] The effects of various self-crosslinked polymeric vesicles
loaded with different drugs on ovarian cancer-bearing mice were
studied by the similar experimental methods described above. The
results are shown in Table 4.
TABLE-US-00004 TABLE 4 Antitumor results in vitro or in vivo of the
drug-loaded self-crosslinked polymeric vesicles against ovarian
cancer Ovarian cancer cell 24 h/pH 7.4 survival release in rate %
Circulatory Drug Maximum survival vitro (%) drug elimination
accumulation tolerated time (day) No 10 mM empty carrying IC.sub.50
half-life in tumor amount subcutaneous orthotopic Polymeric
vesicles GSH GSH vesicles vesicles (.mu.g/mL) (h) (% ID/g) (mg/kg)
model model PEG5k-P (CDC5.8k- 14 78 >90 43.5 8.92 4.79 3.52 120
38 40 co-TMC23k)/ DOX-HCl FA20-CLPs/PEG5k- 21 81 >90 21.6 2.13
4.49 6.54 160 >60 >45 P (CDC5.8k-co- TMC23k)/DOX- HCl
GE11-CLPs/PEG5k- 23 82 >91 16.8 1.92 4.99 8.63 160 >62 >45
P (CDC5.8k-co- TMC23k)/DOX- HCl Transferrin-CLPs/ 24 76 >88 22.7
3.06 5.04 9.02 150 >72 >40 PEG6.5k-P (CDC3.8k- co-LA13.8k)/
DTX
* * * * *